RESUMO
AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.
Assuntos
Antineoplásicos , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Humanos , Antineoplásicos/farmacologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Indóis/farmacologiaRESUMO
Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation. While pathways that counter threats posed by 7,8-dihydro-8-oxoguanine are well characterized, mechanisms protecting against the major adenine oxidation product, 7,8-dihydro-8-oxoadenine (oxoA), are poorly understood. Human DNA polymerases incorporate dGTP or dCTP opposite oxoA, producing mispairs that can cause AâC or AâG mutations. oxoA also perturbs the activity of enzymes acting on DNA and causes interstrand crosslinks. To inform mechanisms for oxoA repair, we characterized oxoA excision by human thymine DNA glycosylase (TDG), an enzyme known to remove modified pyrimidines, including deaminated and oxidized forms of cytosine and 5-methylcystosine. Strikingly, TDG excises oxoA from Gâ oxoA, Aâ oxoA, or Câ oxoA pairs much more rapidly than it acts on the established pyrimidine substrates, whereas it exhibits comparable activity for Tâ oxoA and pyrimidine substrates. The oxoA activity depends strongly on base pairing and is 370-fold higher for Gâ oxoA versus Tâ oxoA pairs. The intrinsically disordered regions of TDG contribute minimally to oxoA excision, whereas two conserved residues (N140 and N191) are catalytically essential. Escherichia coli mismatch-specific uracil DNA-glycosylase lacks significant oxoA activity, exhibiting excision rates 4 to 5 orders of magnitude below that of its ortholog, TDG. Our results reveal oxoA as an unexpectedly efficient purine substrate for TDG and underscore the large evolutionary divergence of TDG and mismatch-specific uracil DNA-glycosylase.
Assuntos
Timina DNA Glicosilase , Humanos , Timina DNA Glicosilase/metabolismo , Reparo do DNA , Adenina/metabolismo , DNA/metabolismo , Escherichia coli/metabolismo , Uracila/metabolismo , Timina , Especificidade por SubstratoRESUMO
In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.
Assuntos
Ilhas de CpG , Desmetilação do DNA , Timina DNA Glicosilase/química , Timina DNA Glicosilase/metabolismo , 5-Metilcitosina/análogos & derivados , Citosina/análogos & derivados , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Epigênese Genética , Humanos , Oxirredução , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Timina DNA Glicosilase/genéticaRESUMO
5-Methylcytosine (mC) is an epigenetic mark that is written by methyltransferases, erased through passive and active mechanisms, and impacts transcription, development, diseases including cancer, and aging. Active DNA demethylation involves TET-mediated stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), or 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and subsequent base excision repair. Many elements of this essential process are poorly defined, including TDG excision of caC. To address this problem, we solved high-resolution structures of human TDG bound to DNA with cadC (5-carboxyl-2'-deoxycytidine) flipped into its active site. The structures unveil detailed enzyme-substrate interactions that mediate recognition and removal of caC, many involving water molecules. Importantly, two water molecules contact a carboxylate oxygen of caC and are poised to facilitate acid-catalyzed caC excision. Moreover, a substrate-dependent conformational change in TDG modulates the hydrogen bond interactions for one of these waters, enabling productive interaction with caC. An Asn residue (N191) that is critical for caC excision is found to contact N3 and N4 of caC, suggesting a mechanism for acid-catalyzed base excision that features an N3-protonated form of caC but would be ineffective for C, mC, or hmC. We also investigated another Asn residue (N140) that is catalytically essential and strictly conserved in the TDG-MUG enzyme family. A structure of N140A-TDG bound to cadC DNA provides the first high-resolution insight into how enzyme-substrate interactions, including water molecules, are impacted by depleting the conserved Asn, informing its role in binding and addition of the nucleophilic water molecule.
Assuntos
Citosina/análogos & derivados , Timina DNA Glicosilase/metabolismo , Citosina/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Timina DNA Glicosilase/químicaRESUMO
MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G(o)) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.
Assuntos
DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Sítios de Ligação , Biocatálise , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Glicosilases/química , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Ligação Proteica , Estabilidade ProteicaRESUMO
5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), and potentially 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair. Here, we investigate the mechanism for TDG excision of fC and caC. We find that 5-carboxyl-2'-deoxycytidine ionizes with pK(a) values of 4.28 (N3) and 2.45 (carboxyl), confirming that caC exists as a monoanion at physiological pH. Calculations do not support the proposal that G·fC and G·caC base pairs adopt a wobble structure that is recognized by TDG. Previous studies show that N-glycosidic bond hydrolysis follows a stepwise (S(N)1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, that is, leaving group quality of the target base. Calculations here show that fC and the neutral tautomers of caC are acidic relative to other TDG substrates, but the caC monoanion exhibits poor acidity and likely resists TDG excision. While fC activity is independent of pH, caC excision is acid-catalyzed, and the pH profile indicates that caC ionizes in the enzyme-substrate complex with an apparent pKa of 5.8, likely at N3. Mutational analysis reveals that Asn191 is essential for excision of caC but dispensable for fC activity, indicating that N191 may stabilize N3-protonated forms of caC to facilitate acid catalysis and suggesting that N191A-TDG could potentially be useful for studying DNA demethylation in cells.
Assuntos
Citosina/análogos & derivados , DNA/metabolismo , Timina DNA Glicosilase/metabolismo , Citosina/química , Citosina/metabolismo , DNA/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Timina DNA Glicosilase/químicaRESUMO
Apurinic/apyrimidinic endonuclease 1 (APE1) is the predominant AP site repair enzyme in mammals. APE1 also maintains 3'-5' exonuclease and 3'-repair activities, and regulates transcription factor DNA binding through its REF-1 function. Since complete or severe APE1 deficiency leads to embryonic lethality and cell death, it has been hypothesized that APE1 protein variants with slightly impaired function will contribute to disease etiology. Our data indicate that except for the endometrial cancer-associated APE1 variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit normal thermodynamic stability of protein folding; abasic endonuclease, 3'-5' exonuclease and REF-1 activities; coordination during the early steps of base excision repair; and intracellular distribution when expressed exogenously in HeLa cells. The R237C mutant displayed reduced AP-DNA complex stability, 3'-5' exonuclease activity and 3'-damage processing. Re-sequencing of the exonic regions of APE1 uncovered no novel amino acid substitutions in the 60 cancer cell lines of the NCI-60 panel, or in HeLa or T98G cancer cell lines; only the common D148E and Q51H variants were observed. Our results indicate that APE1 missense mutations are seemingly rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Neoplasias/metabolismo , Substituição de Aminoácidos/genética , Linhagem Celular Tumoral , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Células HeLa , Humanos , Mutação , Mutação de Sentido Incorreto , Neoplasias/genética , Especificidade por SubstratoRESUMO
DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease.
Assuntos
Domínio Catalítico/fisiologia , Reparo do DNA/fisiologia , Timina DNA Glicosilase/química , Timina DNA Glicosilase/metabolismo , Água/química , 5-Metilcitosina/metabolismo , Ilhas de CpG/genética , Cristalografia , Ativação Enzimática/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Mutagênese/fisiologia , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Estrutura Terciária de Proteína/fisiologia , Especificidade por Substrato , Timina/metabolismo , Timina DNA Glicosilase/genética , Uracila/metabolismo , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G · U activity, but its role in G · T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G · U mispairs and abasic (G · AP) sites, and somewhat less tightly G · T mispairs. Kinetics experiments show 1:1 binding provides full G · T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G · T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G · U or G · T lesions.
Assuntos
Pareamento Incorreto de Bases , DNA/metabolismo , Timina DNA Glicosilase/metabolismo , Ilhas de CpG , DNA/química , Dano ao DNA , Reparo do DNA , Cinética , Ligação Proteica , Timina DNA Glicosilase/químicaRESUMO
Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5' to an abasic site, giving a 3'-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and (13)C(beta)) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Isótopos de Carbono , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Humanos , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular/métodos , Probabilidade , Dobramento de Proteína , SoftwareRESUMO
Cytosine methylation at CpG dinucleotides produces m(5)CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m(5)C deamination yields mutagenic G.T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G.T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G.C pair. hTDG is inactive against normal A.T pairs, and is most effective for G.T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDG(cat)) in complex with abasic DNA, at 2.8 A resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDG(cat) can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.
Assuntos
Pareamento Incorreto de Bases , DNA/metabolismo , Timina DNA Glicosilase/química , Timina DNA Glicosilase/metabolismo , Pareamento de Bases , Sequência de Bases , Ilhas de CpG/genética , Cristalografia por Raios X , DNA/genética , Dimerização , Guanina/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Especificidade por Substrato , TermodinâmicaRESUMO
During reverse transcription, an RNA polypurine tract (PPT) resists digestion by reverse transcriptase (RT) and primes plus-strand DNA synthesis. In this issue of Chemistry & Biology, Yi-Brunozzi et al. (2008) report structural studies of PPTs, illuminating how they are recognized by RT.
Assuntos
DNA Viral/química , Nucleotídeos de Purina/química , RNA Viral/química , Composição de Bases , Sequência de Bases , Sítios de Ligação , DNA Viral/genética , DNA Viral/metabolismo , Transcriptase Reversa do HIV/metabolismo , Nucleotídeos de Purina/genética , Nucleotídeos de Purina/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Thymine DNA glycosylase (TDG) excises thymine from G.T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG.X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG.FU, CpG.ClU, and CpG.BrU than for CpG.T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k(max)) for G.X substrates depends significantly on the 5' base pair. For example, k(max) decreases by 6-, 11-, and 82-fold for TpG.ClU, GpG.ClU, and ApG.ClU, respectively, as compared with CpG.ClU. For the other G.X substrates, the 5'-neighbor effects have a similar trend but vary in magnitude. The activity for G.FU, G.ClU, and G.BrU, with any 5'-flanking pair, meets and in most cases significantly exceeds the CpG.T activity. Strikingly, human TDG activity is reduced 10(2.3)-10(4.3)-fold for A.X relative to G.X pairs and reduced further for A.X pairs with a 5' pair other than C.G. The effect of altering the 5' pair and/or the opposing base (G.X versus A.X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.